1. Quantification of tyrosine, serine and threonine phosphorylation in the TLR signaling pathways. The TLRs are a family of pathogen recognition receptors that alert the host to the presence of pathogens by recognizing molecular signatures, termed pathogen-associated molecular patterns (PAMPs). These sensors act as the first step in the induction of protective innate and adaptive immune responses. There are 11 human TLR homologues and they are each activated by one or more PAMP ligands. TLRs are all transmembrane proteins and their signaling is mediated by association of their internal domains with intracellular components. Classically, the TLR signaling cascade involves the myeloid differentiation primary response gene 88 (MyD88), interleukin-1 receptor-activated kinase (IRAK), and tumor-necrosis factor receptor-associated factor 6 (TRAF6), leading to the activation of Nuclear Factor kappaB (NF-kB). Among the most important genes to be regulated by TLR signaling are those encoding cytokines. Given the key role of cytokines in the orchestration of the inflammatory response, mechanisms of modulating their production has garnered substantial interest, in particular in the area of the development of therapies for the treatment of chronic inflammatory diseases. A clearer understanding of the TLR pathway leading to the cytokine production is required for a successful pharmacological intervention. We are using a well-characterized cell line of murine macrophages and stimulation with TLR ligands to decipher the temporal dynamics of protein phosphorylation in the TLR signaling cascade. We have obtained quantitative data from cells stimulated with LPS (TLR4 ligand), P3C (TLR2 ligand) and R848 (TLR7 ligand) at 6 different time points, analyzed the data and performed the correlation analysis, and identified candidate pathways and proteins to follow up in biological validation experiments. We are preparing a manuscript on the phosphorylation dynamics in the three TLR signaling pathways. We are optimizing another relative quantification method based on post-processing peptide labeling - the dimethylation method, which should be extremely useful for the proteomic and phosphoproteomic studies of the primary cells and tissues not amenable to the SILAC protocol. The method is being tested on the macrophage lysates and will be applied to the studies of the TLR phosphoproteome in primary cells. 2.T-cell receptor signaling - quantification of tyrosine phosphorylation. Recognition of foreign antigens by T lymphocytes is an important step in the initiation of the immune response. Unlike growth factor receptors, the TCR doesn't have intrinsic enzymatic activity. Upon foreign antigen recognition via MHC/TCR, Src family phosphotyrosine kinase (PTK) activity results in immunoreceptor tyrosine-based activation motif (ITAM) phosphorylation and recruitment of ZAP-70 PTK. This allows the TCR complex to function as an active PTK. ZAP-70 phosphorylates adaptor proteins such as LAT and SLP-76 results in activation of phospholipase C (PLC) leading to Ras/Raf1/ERK activation and Ca++ flux. We use T lymphocytes isolated from the 5C.C7 mouse strain and activated in vitro with the antigen presenting cells (line P13.9) as the experimental model system. We have obtained initial qualitative data in this project. 3. Analysis of post-translational modifications of CENP-A (collaboration with Yamini Dalal, NCI): In eukaryotes DNA is packaged into chromatin by essential histone proteins. Specialized histone variants such as centromere-specific histone H3 (CENP-A) provide a structural and epigenetic basis for chromosome segregation by marking centromeres. To maintain centromere parity after replication, CENP-A must segregate equally to nascent daughter DNA strands. How cells prevent unequal distribution of CENP-A to daughter strands after replication fork passage is unknown. We have characterized novel modifications within the histone fold domains of CENP-A and H4 that occur at G1-S cell cycle transition, which coincide with loss of the chaperone HJURP binding, suggesting a mechanistic basis for CENP-A structural conversion. We have established that accurate post-translational modification mapping is possible from minute amounts of protein, using ultra-sensitive mass spectrometry, and bioinformatic tools combined with careful manual spectra validation. We are continuing the detailed characterization of the modification states of CENP-A dependent on the cell cycle stage. Time-resolved, stimulus-dependent quantitative changes of phosphorylation of individual sites are being obtained using sensitive nanospray-based mass spectrometry (LTQ Orbitrap Velos from Thermo) combined with Single Reaction Monitoring. The dynamic modification of selected individual phosphorylation sites is being confirmed by Western blotting.